Fig. 2
From: Can-Seq: a PCR and DNA sequencing strategy for identifying new alleles of known and candidate genes
The Can-Seq workflow. Bulk DNA is prepared from leaf tissue of up to 23 independent mutants. Candidate gene PCR amplicons generated from this template are then combined in equimolar ratios and deep sequenced. Bioinformatic analysis using the Can-Seq script (https://github.com/Carroll-Lab/can_seq) allows for identification of C to T and G to A substitutions at frequencies above an arbitrarily set threshold of 0.75%; the expected frequency for a homozygous candidate mutation in a bulk of 23 independent mutants is 1 in 23 or ~ 4%. The individual mutant containing the candidate mutation is identified via allele-specific PCR assays. Complementation tests involving crosses between independent mutants carrying candidate mutations in the same gene can be used to resolve whether the EMS-induced nucleotide variant detected by Can-Seq is the causative mutation